Low density lipoprotein (hereinafter, referred to as “LDL”) plays a major role in cholesterol transport in blood, and particularly, cholesterol deposited on the walls of blood vessels in atherosclerosis is mainly derived from LDL. An increase in LDL cholesterol is a major risk factor of arteriosclerotic diseases, and its selective quantification is clinically useful. The total cholesterol measurement involves measurement of cholesterol in all lipoproteins such as chylomicron (CM), very low density lipoprotein (VLDL), LDL, and high density lipoprotein (HDL) and is still a main item of lipid test.
Conventional methods for quantifying LDL cholesterol include a method of quantification from two operations of fractionation and cholesterol determination and a method of quantification by calculation based on the values of total cholesterol, HDL cholesterol, and triglycerides according to the Friedewald equation.
For fractionation, there are methods such as ultracentrifugation, precipitation, and immunological technique. These methods require a process for treatment of a sample by ultracentrifugation or filtration and have been difficult to be widely used at laboratory testing sites in view of convenience and cost effectiveness. The calculation method based on the Friedwald equation is limited in its use and associated with accuracy problem because individual differences are not taken into consideration.
Recently, a method of quantification for LDL cholesterol that does not require fractionation has been reported (JP-Patent Publication (Kokai) No. 11-318496 A (1999)) and is currently being used at testing sites as a laboratory testing reagent. This method comprises a first step in which cholesterol in lipoproteins other than LDL in a sample is selectively eliminated (“eliminated” means that ester type cholesterol is degraded and the degradation product is made undetectable in a second step) and a second step in which LDL cholesterol is quantified.
Despite the fact that the reagent for LDL cholesterol measurement described above is a clinically useful reagent, the conventional measurement of total cholesterol is widely performed, and the reagent has not come into wide use because of the reason that LDL cholesterol levels can be determined using the Friedewald equation and so forth. However, there is a problem in LDL cholesterol levels determined by the Friedewald equation as described above, and accurate measurement of LDL cholesterol levels is clinically significant. Hence, it has been desired to further improve the reagent, thereby allowing the reagent for LDL cholesterol measurement of high clinical significance to become widely used.
On the other hand, a method in which cholesterol in HDL and total cholesterol as well as cholesterol in LDL and total cholesterol are sequentially measured by a single measurement was disclosed (JP-Patent Publication (Kohyo) No. 2003-501630 A). In this method, a sample is put in a tube, allowed to form a complex between non-HDL cholesterol in the sample and an anti-apoB antibody, and measured for lipoprotein in uncomplexed form, namely, HDL. Then the complex is dissociated with a surfactant, and remaining non-HDL cholesterol is enzymically measured. By summing the two measurement values, total cholesterol level is found. In the case of LDL cholesterol, a similar reaction method is used, in which an anti-apoA-I or anti-apoA-II antibody is used rather than the anti-apoB antibody at the time of complex formation. HDL cholesterol, LDL cholesterol, and total cholesterol are conventionally widely measured in medical checkup and the like, and the simultaneous measurement of HDL cholesterol, LDL cholesterol, and total cholesterol has been of significance.
Patent document 1: JP-Patent Publication (Kokai) No. 11-318496 A (1999)
Patent document 2: JP-Patent Publication (Kohyo) No. 2003-501630 A